Pax6 affects Ras-Raf-ERK1/2 in mouse aging brain.

Pax6, a transcription factor and multifunctional protein, changes during aging. It also interacts with regulator proteins involved in cell metabolism and survival signalling pathways including Ras-GAP. Many forms of Ras, Raf and ERK1/2 are known but information on their region-specific expression patterns are unavailable from brain during aging. Therefore, it has been intended to evaluate expressions of Pax6 and forms of Ras, Raf, ERK1/2 in hippocampus, caudate nucleus, amygdale, cerebral cortex, cerebellum and olfactory lobe. Association of Pax6 with Ras, Raf and ERK1/2 was evaluated in co-culture (PC-12, C6-glia, U-87 MG) of neuroglia cell lines. Impacts of Pax6 were evaluated by siRNA mediated knockdown and expression patterns Ras-Raf-Erk1/2. Analysis of activities of Pax6 and impacts of 5'AMP, wild-type and mutant ERK were done by RT-PCR and luciferase reporter assay. Results indicate age-dependent changes of Pax6, Ras, Raf, ERK1/2 in different regions of brain of young and old mice. Erk1/2 shows synergistic activities to Pax6.

Age-dependent alterations in expression and co-localization of Pax6 and Ras-GAP in brain of aging mice.

As the brain ages, the survival and plasticity of neurons and glia are compromised. The data-mining and in silico studies suggest interactions of Pax6 with Ras and binding sites in Ras-GAP promoter. The Pax6 also shows age-dependent alterations. Therefore, it is presumed that Pax6 may be associated with the Ras-GAP, a synaptic protein, either directly or indirectly in brain. The expression, co-localization and interaction of Pax6 and Ras-GAP in different regions of brain of mice during aging were investigated through immunofluorescence assay, co-immunoprecipitation and western blotting, respectively. The co-localization of Pax6 and Ras-GAP were observed in dentate gyrus (DG) and sub-granular zone (SGZ) of hippocampus, in glomerular (GlLa) and mitral cells (MiCe) of olfactory lobe, granular cells (GrCe), Purkinje cell (PuCe) and molecular cell layer (MoLa) of cerebellum, internal plexiform layer (InPl), molecular layer (MoLa) of cerebral cortex and in intercalated cells of amygdala (ITC), caudate nucleus regions in brain of aging mice. The expression of Pax6 and Ras-GAP was altered in hippocampus, amygdala, caudate nucleus, olfactory lobe, cerebral cortex and cerebellum from young to old mice. The Pax6 interacts with Ras-GAP in brain of mice. Results indicate impact of Pax6 on Ras-GAP-mediated activities of synapses, learning and memory, emotions and fear as well as motor functions. Alterations in expression and co-localization of Pax6 and Ras-GAP during aging may be responsible for age-associated compromised survival and plasticity of neurons and glia.

Post-donation telephonic interview of blood donors providing an insight into delayed adverse reactions: First attempt in India.

BACKGROUND: Blood donor experiences both immediate adverse reactions (IAR) and delayed adverse reactions (DAR). With limited published data available on the incidence of DAR, a study was conducted to estimate incidence and profile of DAR through telephonic interview. MATERIALS AND METHODS: Study was conducted over a 45-day period for consecutive volunteer whole blood donations at tertiary care hospital. Donors were divided into first-time, repeat and regular and were monitored for IAR. They were given written copy of post-donation advice. Donors were contacted telephonically three weeks post-donation and enquired about general wellbeing and specific DAR in accordance with a standard n international (International Society of Blood Transfusion) standard format. RESULTS: Donors participated in the study of which 1.6% donors experienced an IAR. Much larger number reported DAR (10.3% vs.1.6% p<0.0001). Further, DAR was presented as a variegated profile with bruise, painful arms and fatigue being the commonest. DARs were more common in females than males (25% vs. 10.3%, p<0.02). Localized DAR like bruise and painful arms were more common in younger donors (age <50 years) whereas systemic DAR like fatigue was common in older donors (>50 years). First time (12.3%) and repeat donors (13.5%) had similar frequency of DAR but were lower among regular donors (6.7%). CONCLUSION: DARs are more common than IAR and are of different profile. Post-donation interview has provided an insight into donor experiences and can be used as a valuable tool in donor hemovigilance.

Pax6 influences expression patterns of genes involved in neuro- degeneration.

BACKGROUND: Pax6, a highly conserved multifunctional transcription factor, has been critical for neurogenesis and neuronal plasticity. It is presumed that if level of Pax6 approaches either low or null, critical genes responsible for maintaining functional status of neurons or glia would be modulated. PURPOSE: Therefore, it has been intended to explore possibility of either direct or indirect influence of Pax6 in neurodegeneration. METHODS: The cell lines having origin of murine embryonic fibroblast (Pax6-non expressing, NIH3T3-cell line), murine neuroblastoma (Pax6-expressing brain-derived, Neuro-2a-cell line), and human glioblastoma-astrocytoma (U87MG) were cultured and maintained in a CO2 incubator at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum. The knockdown of endogenous Pax6 in Neuro-2a cells was achieved through siRNA based gene knock-down approach. The efficiency and validation of knock-down was done by real time PCR. The knock-down of Pax6 was successfully achieved. RESULTS: The levels of expression of transcripts of some of the proposed putative markers of neurodegeneration like Pax6, S100ß, GFAP, BDNF, NGN2, p73α, p73δ, LDH, SOD, and Catalase were analyzed in Pax6 knockdown condition for analysis of role of Pax6 in neurodegeneration. Since the Pax6 has been proposed to bind to promoter sequences of catalase, and catalase suppresses TGFß, relative lower levels of catalase in Neuro-2a and U-87MG as compared to NIH-3T3 indicates a possible progressive dominant negative impact of Pax6. However, presence of SOD and LDH indicates alternative protective mechanism. CONCLUSION: Presence of BDNF and TGFß indicates association between them in glioblastoma-astrocytoma. Therefore, Pax6 seems to be involved directly with p53 and TGFß mediated pathways and indirectly with redox-sensitive pathway regulation. The neurodegenerative markers S100ß, GFAP, BDNF, NGN2, p73α, p73δ, observed downregulated in Pax6 knockdown condition suggest Pax6-mediated regulation of these markers. Observations enlighten Pax6-mediated influences on cascades of genes involved in growth, differentiation and maturation of neurons and glia.

Triazino indole-quinoline hybrid: a novel approach to antileishmanial agents.

A novel series of 1,2,4-triazino-[5,6b]indole-3-thione covalently linked to 7-chloro-4-aminoquinoline have been synthesized and evaluated for their in vitro activity against extracellular promastigote and intracellular amastigote form of Leishmania donovani. Among all tested compounds, compounds 7a and 7b were found to be the most active with IC50 values 1.11, 0.36µM and selectivity index (SI) values 67, >1111, respectively, against amastigote form of L. donovani which is several folds more potent than the standard drugs, miltefosine (IC50=8.10µM, SI=7) and sodium stibo-gluconate (IC50=54.60µM, SI⩾7).

A natural product inspired hybrid approach towards the synthesis of novel pentamidine based scaffolds as potential anti-parasitic agents.

A natural product inspired molecular hybridization approach led us to a series of novel pentamidine based pyrimidine and chalcone scaffolds. All the hybrids were evaluated for their anti-leishmanial potential. Most of the screened compounds have showed significant in vitro anti-leishmanial activity with less cytotoxicity in comparison to the standard drugs (pentamidine, sodium stibogluconate, and miltefosine). Additionally, anti-malarial screening of these compounds was also done and four compounds have shown superior activity against chloroquine resistance strain (K1) of Plasmodium falciparum.